This paper reports an in vitro system for studying generalized genetic recombination. The system uses extracts from Escherichia coli as a source of enzymes and plasmid DNA molecules as substrates. Unit-size plasmid DNA rings are converted into genomes fused at a region of DNA homology at a frequency of about 5-10% over a period of hours. That the fused structures are the result of recombination is supported by two lines of evidence. When two partially homologous plasmids of different sizes are used as substrates for the in vitro system, intermediates containing one plasmid of each size are obtained. Furthermore, fused structures are not formed with high efficiency in extracts from recombination-deficient (Rec A(-)) cells.DNA synthesis does not appear to be required for the formation of the recombination intermediates; it is possible to omit DNA precursors from the reaction mixture and, furthermore, to develop the fused structures even in the presence of chaintermininating dideoxynucleoside triphosphates. The structures formed in vitro have the basic properties of recombination intermediates previously recovered from intact cells. That is, two genomes are demonstrably fused at a region of homology. However, in one way the molecules formed in vitro have a property less frequently observed in vivo-the fused genomes often appear to be connected over an extended region of homology ranging up to several hundred base pairs in length. This extended region of pairing may indicate the presence of two crossover connections very close together and, as will be discussed, may provide an insight into the mechanism by which the recombination intermediate is formed.